Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Summary

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Abstract

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial. 

Introduction

Scanning electron microscope (SEM) ultrastructural analysis and intracellular imaging supplement light microscopy for three-dimensional profiling of prokaryotes, plants, and animals. The high spatial resolution of an SEM makes it one of the most versatile and powerful techniques available for the examination of microstructural characteristics of specimens at the nanometer to micrometer scale. Desiccated specimens are resolved to compositional and topographical structures with intense detail, which provides the foundation for developing valid conclusions about functional relationships^1,2,

Protocol

NOTE: Painted turtle (Chrysemys picta) eggs used in this study were collected during the nesting season of May through June 2015-16 from Rice Creek Field Station, Oswego New York with permission obtained from the New York State Department of Environmental Conservation (DEC).

1. Chemical drying method to process embryos for SEM

1. Collect turtle eggs from field sites during the nesting season. Prepare the incubation chambers in advance, made of plastic boxes with lids (L x W x.......

Discussion

In our study, different fixation agents, dehydration and drying methods were tested to prepare three different delicate biological samples for SEM imaging: embryos, eggshells, and fungal cultures. SEM is commonly used for surface analysis, so fixative penetration is less concerning, but it must be understood that poorly fixed internal structures will cause inward shrinking or/and collapsed surface structures. Extended fixation time should also be considered for larger tissue samples, replacing the fixative solution a few.......

Materials

Name	Company	Catalog Number	Comments
Agar	Fischer Scientific	S25127A	for slide cultures
Aluminum pin stub	Tedpella	16111	12.7 mm x 8 mm
BD Difco Dehydrated Culture Media: Potato Dextrose Agar	BD 213400	DF0013-17-6	Media for isolation and cultivation of Fungi, yeast and molds
Chloramphenicol	Fischer BioReagents	BP904-100	Antibiotic for media
Coarse Vermiculite	Greenhouse Megastore	SO-VER-12	bedding medium
Clear 12- well plate	Corning	07-201-589	for fixing embryo
Coverslips	Fischer Scientific	S17525B	for slide culture
Critical Point Dryer	Quorum CPD	EMS850	critical point drying
Culture dishes	Fischer Scientific	08 747B	DISH PETRI 100X10MM 12/PK
Ethanol	Fischer Scientific	A406P 4	dehydration agent
Forceps- Aquarius Tweezers	Tedpella	5804	style 4, length 108mm, widh x thickness 0.017 x 0.17 mm
Glutaraldehyde	Fischer Scientific	G151-1	fixative
Gold target for sputter coater	DENTON VACUUM	TAR001-0158	Gold Target, 2.375″ D X .002″
Hexamethyldisilazana	Fischer Scientific	C19479-5000	chemical drying agent
Kim wipes	Kimtech	S-8115	cleaning
Microscope slides	Thermo Scientific	67-762-16	for slide culture
Microscopy Scissors	Tedpella	1327	Double pointed, stainless steel, 100 mm L (3-5/8").
Micro-scissors	Tedpella	1346	Vannas-type, straight, 80mm L
Moria Perforated Embryo Spoon	Fine Science Tools	10370-17	Length 14.5 cm, tip diameter 20 mm, spoon depth 5 mm
Netwell Inserts	Corning	0330B09	15 mm Inserts with 74 µm Mesh Size Polyester Membrane act as handy carriers during specimen processing into different solvents
Paraformaldehyde	Fischer Scientific	T353 500	fixative
Peat moss	Walmart- Miracle Gro	551705263	bedding medium
PELCO tabs double stick carbon conductive tape	Tedpella	5000	12 mm OD
Sputter coater	DENTON VACUUM	DESK V	thin metal coating
SEM	JEOL USA	JEOL JSM 6610LV scanning electron scope	electron microscopy